Ror1-specific chimeric antigen receptors (car) with humanized targeting domains

ABSTRACT

The invention relates to chimeric antigen receptors (CAR) with a humanized targeting domain specific to the antigen ROR1. The invention encompasses the polynucleotides, vectors encoding said CARs and the isolated cells expressing them at their surface, in particularly for their use in immunotherapy.

FIELD OF THE INVENTION

The invention relates to chimeric antigen receptors (CAR) with a humanized targeting domain specific to the antigen ROR1. The invention encompasses the polynucleotides, vectors encoding said CARs and the isolated cells expressing them at their surface, in particularly for their use in immunotherapy.

BACKGROUND OF THE INVENTION

The adoptive transfer of genetically modified T cells that express a T cell receptor or a chimeric antigen receptor (CAR) specific for a tumor-associated antigen is emerging as an effective modality for cancer therapy [1-5]. CARs are synthetic receptors most often constructed by linking a single chain variable fragment (scFV) of a monoclonal antibody (mAb) specific for a tumor cell surface molecule to a transmembrane domain, one or more intracellular costimulatory signaling modules, and CD3ζ [6-8]. CAR-modified T-cells confer non-MHC restricted recognition of tumor cells, and durable responses have been reported in patients with B-cell malignancies after treatment with autologous T-cells modified with CARs specific for the B-cell lineage restricted CD19 molecule. The major toxicities in these patients were related to tumor lysis, cytokine release, and prolonged depletion of normal B-lymphocytes [1-3, 5, 9]. A challenge in the field is to identify and validate receptor constructs specific for molecules that are expressed on a greater number of malignancies including common epithelial tumors, and that are restricted in their expression to malignant and not normal cells.

We have been investigating the receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a candidate for immunotherapy with CAR-modified T-cells. ROR1 is a 120-kDa glycoprotein containing extracellular immunoglobulin (Ig)-like, Frizzled, and Kringle domains. ROR1 is expressed during embryogenesis but absent from normal adult tissues, apart from a subset of immature B-cell precursors, and low-level expression on adipocytes [10, 11]. ROR1 was first shown to be expressed in B-cell chronic lymphocytic leukemia (B-CLL) by transcriptional profiling [12, 13], and was subsequently identified on the surface of many cancers including mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) with a t(1;19) chromosome translocation, and a subset of lung, breast, colon, pancreas, renal, and ovarian cancers [14-21]. In both lung adenocarcinoma and t(1;19) ALL, ROR1 cooperates in oncogenic signaling, and knockdown of ROR1 with siRNA exposed a critical role for this molecule in maintaining tumor cell survival [15, 18, 22, 23]. WO 2016/115559 relates to antibodies and chimeric antigen receptors specific for ROR1.

Clinical trials with CD19 CARs have demonstrated that a paramount requirement for therapeutic effficacy is engraftment, in vivo proliferation and persistence of CAR T cells after adoptive transfer [24-26] (Clinical trial IDs: NCT02167360, NCT02030847 NCT01865617). In responding patients, CAR T cells undergo substantial proliferation, to a point where they comprise a substantial proportion of the patient's T-cell repertoire. Non-responding patients by contrast have an insufficient CAR T cell engraftment or the CAR T cell graft is rejected early after adoptive transfer. CAR T cell proliferation is an ‘advanced’ effector function, requiring optimal binding of the CAR targeting domain to the respective antigen. Therefore, to maximize the potential of CAR T cell therapy it is important to chose a CAR that exhibits strong antigen binding properties and also mediates high CAR T cell expansion and long-term survival.

All CARs that are currently used in the clinic contain scFv targeting domains that are derived from murine antibodies and the majoritiy of CARs that are in preclinical development also use targeting domains of ‘foreign’ origin. It has been noted that these ‘foreign’ targeting domains contain immunogenic epitopes that are recognized by the patient's immune system and elicit cellular or humoral immune responses that eventually mediate CAR-T cell rejection; see [27], [28] and [29]. Reference [30] describes the use of targeting domains from human antibodies.

In summary, there is a need in the art for improved CAR T cell constructs and related products and methods that can be used for therapies such as immunotherapies.

DESCRIPTION OF THE INVENTION

The present invention relates to humanized binding domains based on the known anti-ROR1 antibodies R11, R12 and 2A2 and to uses thereof for the construction of CARs and CAR engineered T cells. See, for instance, reference [14] for a description of the 2A2 antibody, which is hereby incorporated by reference in its entirety for all purposes; and reference [37] for a description of the R11/R12 antibodies, which is hereby incorporated by reference in its entirety for all purposes. The CARs according to the present invention have a higher degree of “human-ness” of the humanized R11, R12 and 2A2 binding domains, as opposed to the rabbit (R11 & R12) and mouse (2A2) binding domains. According to the invention, these CARs and CAR engineered T cells are expected to exhibit lower immunogenicity in clinical use in patients, as opposed to the rabbit (R11 & R12) and mouse (2A2) binding domains.

According to the invention, humanization can be carried out by any method that is known in the art. As a non-limiting example, humanization of the VH and VL domains of R11, R12 and 2A2 anti-ROR1 monoclonal antibodies has been performed by CDR grafting and selection of best binders for the ROR-1 target by Transpo-mAb Display. This method has been described for the R11 and 2A2 antibodies in reference [31], which is hereby incorporated by reference in its entirety for all purposes. According to the invention, recombinant mammalian cells expressing CARs such as CAR T cells can be produced to express CARs with the humanized anti-ROR1 binding domains of monoclonal anti-ROR-1 antibodies R11, R12 and 2A2.

Humanization of ROR1-specific CARs is different from the known clinical approaches which rely on non-humanized ROR1-specific CARs. The present inventors have now surprisingly shown that humanized ROR1-specific CARs have higher functionality than their non-humanized counterparts. This advantage was unexpected, because humanization is thought to decrease rather than increase affinity.

Furthermore, the inventors have surprisingly shown that humanized CARs with advantageous functional properties can be produced according to the invention. For instance, CAR T cells according to the invention are capable of target cell lysis and exhibit strong proliferation upon stimulation with lethally irradiated ROR1-expressing target cells.

Accordingly, the present invention provides the following preferred embodiments:

-   1. A ROR1-specific CAR comprising:     -   a humanized targeting domain obtainable by humanization of a         ROR1-binding fragment of a monoclonal antibody capable of         binding to ROR1, wherein said monoclonal antibody is selected         from the group consisting of the monoclonal antibodies R11, R12         and 2A2. -   2. The ROR1-specific CAR according to item 1, wherein said     monoclonal antibody is selected from the group consisting of the     monoclonal antibodies R12 and 2A2. -   3. A ROR1-specific CAR comprising a humanized targeting domain     capable of binding to ROR1, wherein the humanized targeting domain     comprises, preferably in an N- to C-terminal order:     -   a) an antibody heavy chain variable domain amino acid sequence         selected from the group consisting of:         -   a1) the amino acid sequence of SEQ ID No: 1 or an amino acid             sequence at least 90% identical thereto;         -   a2) the amino acid sequence of SEQ ID No: 3 or an amino acid             sequence at least 90% identical thereto; or         -   a3) the amino acid sequence of SEQ ID No: 5 or an amino acid             sequence at least 90% identical thereto;     -   and     -   b) an antibody light chain variable domain amino acid sequence         selected from the group consisting of:         -   b1) the amino acid sequence of SEQ ID No: 2 or an amino acid             sequence at least 90% identical thereto;         -   b2) the amino acid sequence of SEQ ID No: 4 or an amino acid             sequence at least 90% identical thereto; or         -   b3) the amino acid sequence of SEQ ID No: 6 or an amino acid             sequence at least 90% identical thereto. -   4. The ROR1-specific CAR according to item 3, wherein the humanized     targeting domain comprises the antibody heavy chain variable domain     amino acid sequence of SEQ ID No: 1 or an amino acid sequence at     least 90% identical thereto and the antibody light chain variable     domain amino acid sequence of SEQ ID No: 2 or an amino acid sequence     at least 90% identical thereto. -   5. The ROR1-specific CAR according to item 4, wherein the humanized     targeting domain comprises the antibody heavy chain variable domain     amino acid sequence of SEQ ID No: 1 and the antibody light chain     variable domain amino acid sequence of SEQ ID No: 2. -   6. The ROR1-specific CAR according to item 5, wherein the humanized     targeting domain consists of the following sequences in an N- to     C-terminal order: the antibody heavy chain variable domain amino     acid sequence of SEQ ID No: 1, an amino acid linker sequence which     is preferably the amino acid sequence of SEQ ID No: 8, and the     antibody light chain variable domain amino acid sequence of SEQ ID     No: 2. -   7. The ROR1-specific CAR according to item 3, wherein the humanized     targeting domain comprises the antibody heavy chain variable domain     amino acid sequence of SEQ ID No: 3 or an amino acid sequence at     least 90% identical thereto and the antibody light chain variable     domain amino acid sequence of SEQ ID No: 4 or an amino acid sequence     at least 90% identical thereto. -   8. The ROR1-specific CAR according to item 7, wherein the humanized     targeting domain comprises the antibody heavy chain variable domain     amino acid sequence of SEQ ID No: 3 and the antibody light chain     variable domain amino acid sequence of SEQ ID No: 4. -   9. The ROR1-specific CAR according to item 8, wherein the humanized     targeting domain consists of the following sequences in an N- to     C-terminal order: the antibody heavy chain variable domain amino     acid sequence of SEQ ID No: 3, an amino acid linker sequence which     is preferably the amino acid sequence of SEQ ID No: 8, and the     antibody light chain variable domain amino acid sequence of SEQ ID     No: 4. -   10. The ROR1-specific CAR according to item 3, wherein the humanized     targeting domain comprises the antibody heavy chain variable domain     amino acid sequence of SEQ ID No: 5 or an amino acid sequence at     least 90% identical thereto and the antibody light chain variable     domain amino acid sequence of SEQ ID No: 6 or an amino acid sequence     at least 90% identical thereto. -   11. The ROR1-specific CAR according to item 10, wherein the     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 5 and the antibody     light chain variable domain amino acid sequence of SEQ ID No: 6. -   12. The ROR1-specific CAR according to item 11, wherein the     humanized targeting domain consists of the following sequences in an     N- to C-terminal order: the antibody heavy chain variable domain     amino acid sequence of SEQ ID No: 5, an amino acid linker sequence     which is preferably the amino acid sequence of SEQ ID No: 8, and the     antibody light chain variable domain amino acid sequence of SEQ ID     No: 6. -   13. A combination of CARs comprising at least a first and a second     CAR, the combination being ROR1-specific, wherein said first and     said second CAR are present on different polypeptide chains, and     wherein:     -   c) said first CAR comprises a first humanized targeting domain         comprising an antibody heavy chain variable domain amino acid         sequence selected from the group consisting of:         -   c1) the amino acid sequence of SEQ ID No: 1 or an amino acid             sequence at least 90% identical thereto;         -   c2) the amino acid sequence of SEQ ID No: 3 or an amino acid             sequence at least 90% identical thereto; or         -   c3) the amino acid sequence of SEQ ID No: 5 or an amino acid             sequence at least 90% identical thereto; -    and     -   d) said second CAR comprises a second humanized targeting domain         comprising an antibody light chain variable domain amino acid         sequence selected from the group consisting of:         -   d1) the amino acid sequence of SEQ ID No: 2 or an amino acid             sequence at least 90% identical thereto;         -   d2) the amino acid sequence of SEQ ID No: 4 or an amino acid             sequence at least 90% identical thereto; or         -   d3) the amino acid sequence of SEQ ID No: 6 or an amino acid             sequence at least 90% identical thereto. -   14. The combination according to item 13, wherein the first     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 1 or an amino acid     sequence at least 90% identical thereto and the second humanized     targeting domain comprises the antibody light chain variable domain     amino acid sequence of SEQ ID No: 2 or an amino acid sequence at     least 90% identical thereto. -   15. The combination according to item 14, wherein the first     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 1 and the second     humanized targeting domain comprises the antibody light chain     variable domain amino acid sequence of SEQ ID No: 2. -   16. The combination according to item 13, wherein the first     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 3 or an amino acid     sequence at least 90% identical thereto and the second humanized     targeting domain comprises the antibody light chain variable domain     amino acid sequence of SEQ ID No: 4 or an amino acid sequence at     least 90% identical thereto. -   17. The combination according to item 16, wherein the first     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 3 and the second     humanized targeting domain comprises the antibody light chain     variable domain amino acid sequence of SEQ ID No: 4. -   18. The combination according to item 13, wherein the first     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 5 or an amino acid     sequence at least 90% identical thereto and the second humanized     targeting domain comprises the antibody light chain variable domain     amino acid sequence of SEQ ID No: 6 or an amino acid sequence at     least 90% identical thereto. -   19. The combination according to item 18, wherein the first     humanized targeting domain comprises the antibody heavy chain     variable domain amino acid sequence of SEQ ID No: 5 and the second     humanized targeting domain comprises the antibody light chain     variable domain amino acid sequence of SEQ ID No: 6. -   20. The ROR1-specific CAR according to any one of the preceding     items, wherein the CAR further comprises a costimulatory domain     capable of mediating costimulation to immune cells; or the     combination according to any one of the preceding items, wherein at     least the first or the second CAR of the combination, preferably at     least the first and the second CAR of the combination, most     preferably all of the CARs of the combination, further comprise a     costimulatory domain capable of mediating costimulation to immune     cells. -   21. The ROR1-specific CAR or the combination according to item 20,     wherein said costimulatory domain is from 4-1BB, CD28, Ox40, ICOS or     DAP10. -   22. The ROR1-specific CAR or the combination according to item 21,     wherein said costimulatory domain has the amino acid sequence of SEQ     ID No: 12. -   23. The ROR1-specific CAR according to any one of the preceding     items, further comprising a transmembrane polypeptide; or the     combination according to any one of the preceding items, wherein at     least the first or the second CAR of the combination, preferably at     least the first and the second CAR of the combination, most     preferably all of the CARs of the combination, further comprise a     transmembrane polypeptide. -   24. The ROR1-specific CAR or the combination according to item 23,     wherein said transmembrane polypeptide is a transmembrane domain     from CD4, CD8 or CD28. -   25. The ROR1-specific CAR or the combination according to item 24,     wherein said transmembrane domain has the amino acid sequence of SEQ     ID No: 11. -   26. The ROR1-specific CAR according to any one of the preceding     items, further comprising a CAR spacer domain; or the combination     according to any one of the preceding items, wherein at least the     first or the second CAR of the combination, preferably at least the     first and the second CAR of the combination, most preferably all of     the CARs of the combination, further comprise a CAR spacer domain. -   27. The ROR1-specific CAR or the combination according to item 26,     wherein said CAR spacer domain is from CD4, CD8, an FC-receptor, an     immunoglobulin, or an antibody. -   28. The ROR1-specific CAR or the combination according to item 27,     wherein said CAR spacer domain has the amino acid sequence of SEQ ID     No: 9 or SEQ ID No: 10, preferably the amino acid sequence of SEQ ID     No: 9. -   29. The ROR1-specific CAR according to any one of the preceding     items, further comprising a suicide gene product that allows the     selective killing of CAR T cells; or the combination according to     any one of the preceding items, wherein at least the first or the     second CAR of the combination further comprises a suicide gene     product that allows the selective killing of CAR T cells. -   30. The ROR1-specific CAR or the combination according to item 29,     wherein said suicide gene product is iCasp9 or HSV-TK. -   31. The ROR1-specific CAR according to any one of the preceding     items, comprising, in an N- to C-terminal order: i) a signal peptide     for direction into the endoplasmic reticulum, the signal peptide     preferably having the amino acid sequence of SEQ ID No: 7; ii) said     humanized targeting domain; iii) the CAR spacer domain according to     any one of items 26 to 28; iv) the transmembrane polypeptide     according to any one of items 23-25; v) the costimulatory domain     according to any one of items 20-22; vi) a CD3z signaling domain     preferably having the amino acid sequence of SEQ ID No: 13; and     optionally further comprising: vii) a T2A ribosomal skipping     sequence preferably having the amino acid sequence of SEQ ID No:     14; viii) a signal peptide for direction into the endoplasmic     reticulum, the signal peptide preferably having the amino acid     sequence of SEQ ID No: 7; and ix) a detectable marker protein     sequence preferably having the EGFRt amino acid sequence of SEQ ID     No: 15. -   32. The ROR1-specific CAR according to item 31, consisting of said     components i) to ix). -   33. A polynucleotide encoding the ROR1-specific CAR according to any     one of the preceding items. -   34. A recombinant mammalian cell expressing at least one     ROR1-specific CAR according to any one of the preceding items or     expressing the combination of CARs according to any one of the     preceding items. -   35. The recombinant mammalian cell according to item 34, wherein     said cell is an immune cell. -   36. The recombinant mammalian cell according to any one of the     preceding items, wherein said cell is a lymphocyte. -   37. The recombinant mammalian cell according to any one of the     preceding items, wherein said lymphocyte is a B lymphocyte or T     lymphocyte. -   38. The recombinant mammalian cell according to item 34, wherein     said cell is a CD8⁺ killer T cell, a CD4⁺ helper T cell, a naive T     cell, a memory T cell, a central memory T cell, an effector memory T     cell, a memory stem T cell, an invariant T cell, an NKT cell, a     cytokine induced killer T cell, a g/d T cell, a natural killer cell,     a monocyte, a macrophage, a dendritic cell, or a granulocyte. -   39. The recombinant mammalian cell according to any one of the     preceding items, wherein the cell is a human cell. -   40. The recombinant mammalian cell according to any one of the     preceding items, wherein the recombinant mammalian cell is capable     of at least one cell division when cocultured with lethally     irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for     72 h in the absence of exogenous cytokines. -   41. The recombinant mammalian cell according to any one of the     preceding items, wherein the recombinant mammalian cell is capable     of at least two cell divisions when cocultured with lethally     irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for     72 h in the absence of exogenous cytokines. -   42. The recombinant mammalian cell according to any one of the     preceding items, wherein the recombinant mammalian cell is capable     of at least three cell divisions when cocultured with lethally     irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for     72 h in the absence of exogenous cytokines. -   43. The recombinant mammalian cell according to any one of the     preceding items, wherein the recombinant mammalian cell is capable     of at least four cell divisions when cocultured with lethally     irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for     72 h in the absence of exogenous cytokines. -   44. The recombinant mammalian cell according to any one of the     preceding items, wherein the recombinant mammalian cell is capable     of at least five cell divisions when cocultured with lethally     irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for     72 h in the absence of exogenous cytokines. -   45. The recombinant mammalian cell according to any one of the     preceding items, wherein the ROR1-specific CAR or the ROR1-specific     combination of CARs is capable of binding to ROR1 with higher     binding affinity than a respective recombinant mammalian control     cell expressing a respective ROR1-specific CAR or a respective     ROR1-specific combination of CARs where none of the targeting     domains is humanized, and wherein said binding affinity is binding     affinity to fluorescently labelled recombinant aggregated ROR1 as     assessed by flow cytometry analysis. -   46. A method for producing a recombinant mammalian cell according to     any one of the preceding items, the method comprising the steps of:     -   (a) providing a mammalian cell;     -   (b) introducing into said mammalian cell of step (a) at least         one polynucleotide encoding said at least one ROR1-specific CAR         or said combination of CARs; and     -   (c) expressing said at least one ROR1-specific CAR or said         combination of CARs from said at least one polynucleotide in         said cell; -    thereby obtaining said recombinant mammalian cell. -   47. The method of item 46, further comprising coculturing the     recombinant mammalian cell with lethally irradiated ROR1-expressing     target cells at a ratio of 4:1 for 72 h in the absence of exogenous     cytokines. -   48. The method of item 47, wherein at least 1%, preferably at least     2%, more preferably at least 3%, and still more preferably at least     4% of the recombinant mammalian cells undergo at least 5 cell     divisions during the coculturing. -   49. The method according to any one of the preceding items, wherein     said method is an in vitro method. -   50. The method according to any one of the preceding items, wherein     said mammalian cells of step (a) are cells obtained from donors. -   51. The method according to any one of the preceding items, wherein     said mammalian cells of step (a) are cells obtained from patients. -   52. A recombinant mammalian cell according to any one of the     preceding items for use in medicine. -   53. A recombinant mammalian cell according to any one of the     preceding items for use in a method for treating a cancer that     expresses ROR1 in a patient. -   54. The recombinant mammalian cell according to item 53 for the use     according to item 53, wherein the cancer is ROR1-positive leukemia,     mantle cell lymphoma, breast-cancer or lung cancer. -   55. A method for treating a patient in need thereof, the method     comprising administering a recombinant mammalian cell according to     any one of the preceding items to said patient. -   56. The method according to item 55, wherein the method is for     treating a cancer that expresses ROR1. -   57. The method according to item 56, wherein the cancer is     ROR1-positive leukemia, mantle cell lymphoma, breast-cancer or lung     cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid sequences of the humanized 2A2 VH and VL sequences and the complete h2A2 CAR coding sequence

-   -   (A) Humanized 2A2 VH and VL amino acid sequences     -   (B) Complete amino acid sequence of the h2A2 CAR, represented in         an N- to C-terminal order. Note that the asterisk denotes the         end of the amino acid sequence.

FIG. 2: Amino acid sequences of the humanized R11 VH and VL sequences and the complete R11 CAR coding sequence

-   -   (A) Humanized R11 VH and VL amino acid sequences     -   (B) Complete amino acid sequence of the R11 CAR, represented in         an N- to C-terminal order. Note that the asterisk denotes the         end of the amino acid sequence.

FIG. 3: Amino acid sequences of the humanized R12 VH and VL sequences and the complete R12 CAR coding sequence

-   -   (A) Humanized R12 VH and VL amino acid sequences     -   (B) Complete amino acid sequence of the R12 CAR, represented in         an N- to C-terminal order. Note that the asterisk denotes the         end of the amino acid sequence.

FIG. 4: Enrichment and detection of CAR by EGFRt transduction marker

Human CD4 or CD8 positive T cells were transduced with lentiviral vector encoding a humanized or non-humanized ROR1 CAR and subsequently enriched for CAR expressing cells by magnetic activated cell sorting (MACS) making use of the truncated epidermal growth factor receptor (EGFRt) transduction marker. The coding sequence (CDS) for EGFRt is linked to the CAR CDS by a 2A ribosomal skipping sequence and expression of EGFRt can be used as a surrogate marker for CAR expression.

-   -   (A) Flow cytometry plots demonstrating the frequency of         EGFRt-positive CD4⁺ T cells after EGFRt enrichment by MACS.     -   (B) Flow cytometry plots demonstrating the frequency of         EGFRt-positive CD8⁺ T cells after EGFRt enrichment by MACS.

FIG. 5: Cytolytic activity of hROR1 CAR-expressing T cells

-   -   (A) Cytolytic activity of primary human CD8⁺ T cells expressing         the indicated non-humanized or humanized ROR1-specific CARs         against ROR1-positive target cells. K562 is a ROR1-negative         human leukemia cell line that was used as negative control.         K562-ROR1 originates from the same cell line but has been         engineered to stably express ROR1. MDA-MB-231 and A549 are human         breast and lung cancer cell lines that endogenously express         ROR1. All target cell lines were engineered to stably express a         firefly (P. pyralis) luciferase. The specific lysis of the         target cells was calculated based on the intensity of the         luminescence signal after addition of Luciferin to a final         concentration of 150 μg/ml.     -   (B) Summary of the cytolytic activity of human T cells         expressing a non-humanized or humanized ROR1-specific CAR         against two ROR1 positive target cells lines K562-ROR1 and         MDA-MB-231. The data was collected from n=3 independent         experiments. The specific lysis was calculated based on the         luminesce intensity of ffluc-positive target cells after 6 h         incubation with an E:T ratio of 10:1.

FIG. 6: Cytokine secretion of hROR1 CAR-expressing T cells

CD4⁺ or CD8⁺ CAR T cells expressing a non-humanized or humanized ROR-specific CAR were co-cultured with lethally irradiated ROR1-positive target cells at an E:T ratio of 4:1. Concentrations of the effector cytokines IL-2 and IFN-γ were measured by ELISA in the cell culture supernatants after 24 h co-culture.

-   -   (A) Comparison of cytokine secretion from 2A2 and h2A2 CAR T         cells     -   (B) Comparison of cytokine secretion from R11 and hR11 CAR T         cells     -   (C) Comparison of cytokine secretion from R12 and hR12 CAR T         cells

FIG. 7: Proliferation of hROR1 CAR-expressing T cells

Proliferation of CD4⁺ ROR1-specific CAR T cells after stimulation with ROR1-positive target cells at an E:T ratio of 4:1. No exogenous cytokines were added to the culture media and the T cell proliferation was assessed by CFSE dye dilution 72 h after stimulation. For analysis, triplicate wells were pooled and the proliferation of live 7AAD⁻, CD4+ T cells was analyzed.

-   -   (A) CFSE flow cytometry histograms of ROR1 CAR T cells with         humanized (solid line) or non-humanized (dashed line). Grey         filled curves are from vector control T cells (EGFRt).     -   (B) Division indices of indicated ROR1 CAR T cells     -   (C) Table that summarized the percentages of T cells that went         through 0, 1, 2, 3, 4, and 5 cell division cycles.

FIG. 8: Binding of ROR1 protein by hROR1 CAR T cells

Human CD8⁺ T cells expressing non-humanized or humanized ROR1 CARs were collected, washed with PBS, 0.25% FCS and then incubated 10 min in the same buffer containing a final concentration of 5.3 μ/ml AlexaFluor 647-labeled aggregated ROR1 protein and monoclonal αEGFR antibody. Afterwards the cells were washed with PBS, 0.25% FCS and analyzed by flow cytometry.

FIG. 9: In vivo activity of hROR1 CAR T cells

NSG mice were inoculated with ROR1-expressing Jeko-1 mantle cell lymphoma lines and received a treatment of humanized R12 or 2A2 CAR T cells 21 days later.

-   -   (A) Mice were injected with luciferin at day 28 and radiance         signals, which are emitted by ffluc-positive tumor cell, were         detected. Displayed is the change of average radiance per mouse         for the four best responding mice of each group compared to the         baseline signals, which were measured on day 21.     -   (B) Flow cytometry plots showing the frequency of EGFRt-positive         CD4⁺ and CD8⁺ T cells after EGFRt in the bone marrow of mice         that received hR12 or h2A2 ROR1 CAR T cells at day 56.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, immunology, biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. References referred to herein are indicated by a reference number in square brackets (e.g. as “[31]” or as “reference [31]”), which refers to the respective reference in the list of references at the end of the description. In case of conflict, the present specification, including definitions, will prevail over the cited references. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

Antibodies of non-human origin can be humanized by CDR grafting by methods known in the art. The humanization increases the homology of the binding domains to human antibody binding domains (i.e. the humanness), and reduces the immunogenic potential of the humanized antibody in human beings, which in turn is expected to increase the safety and therapeutic application profile in human patients. On the other hand, antibody humanization is often accompanied by a reduction of the binding affinity of the humanized antibody to its antigen, often requiring tedious affinity maturation, see reference [32], which is hereby incorporated by reference in its entirety for all purposes. It has also been experienced that the use of humanized antibody fragments for the generation of a targeting domain of a CAR can result in a lower performance of the CAR in respect to binding to the target antigen and triggering effector functions of the CAR expressing cell.

In the present invention the inventors have fused humanized VH and VL domains, as exemplified in FIGS. 1-3, of antibodies originating from the anti-ROR1-specific antibody clones 2A2 (Mus musculus, WO2010/124188, which is hereby incorporated by reference in its entirety for all purposes), R11 and R12 (Oryctolagus cuniculus, WO2012/075158, which is hereby incorporated by reference in its entirety for all purposes). The humanized VH and VL domains of these antibodies were then used to design single chain variable fragments (scFvs) that were further used for the design of CARs targeting the ROR1 antigen. Gene transfer vectors were created that allow the transduction or transfection of primary human T cells with the humanized CARs and allow the production of a CAR T cell product. The effector functions of the CAR T cells expressing ROR1 CARs with humanized ROR1 targeting domains were compared to their non-humanized counterparts. The humanized ROR1 CARs h2A2 and hR12 have conferred unexpected superior effector functions compared to their non-humanized counterparts.

The present invention describes for the first time the generation of humanized ROR1 CARs and their usage to redirect immune cells for the killing of ROR1 expressing target cells. Unexpectedly, the observed activity of two of the humanized CARs, namely h2A2 and hR12, was higher than the non-humanized forms in functional T cells assays with ROR1-expressing target cells. In contrast, the CAR that was constructed with an scFv targeting domain that originated from the R11 monoclonal antibody showed a comparatively strong reduction of effector functions, thus demonstrating the commonly expectable decline in therapeutic potential.

The finding of the present invention, that humanized ROR1 CARs can mediate antigen-specific effector functions to immune cells that are superior to those of the non-humanized counterparts is unexpected and has, to the inventors' knowledge, not been disclosed in the prior art. This finding is also unexpected, because antibody humanization is often accompanied by a loss of affinity, and/or a reduction of affinity to the target antigen. CARs whose targeting domain originates from such humanized antibodies oftentimes also exhibit lower effector functions and less therapeutic potential. It could thus neither be anticipated nor expected that our humanized ROR1 CARs demonstrate effector functions that are superior to their non-humanized counterparts.

The significantly higher function in combination with the anticipated lower immunogenicity of our novel humanized ROR1 CARs provides a substantial advantage for the clinical application of these CARs, especially, but not limited to, their usage in the context of immunotherapy against cancer.

A “recombinant mammalian cell” according to the invention can be any cell as defined herein. Preferably, a recombinant mammalian cell is an isolated cell. Recombinant mammalian cells according to the invention can be produced in accordance with known pharmaceutical standards. For instance, they can be formulated for administration to humans.

A ROR1-specific CAR or a combination of CARs according to the invention can be any possible form. In a preferred embodiment, the ROR1-specific CAR or combination of CARs is present in an isolated form. In another preferred embodiment the ROR1-specific CAR or combination of CARs according to the invention can be present in a composition, The composition may be a pharmaceutical composition.

Sequence alignments of sequences according to the invention are performed by suitable algorithms, and preferably by using the BLAST algorithm, see references [33, 34], using suitable alignment parameters as known in the art.

As used herein, each occurrence of terms such as “comprising” or “comprises” may optionally be substituted with “consisting of” or “consists of”.

The sequences corresponding to the SEQ IDs referred to herein are indicated in FIGS. 1 to 3 and in the following tables:

GMCSF signal peptide (SEQ ID No: 7) MLLLVTSLLLCELPHPAFLLIP h2A2 heavy chain variable domain (VH) (SEQ ID No: 1) EVQLVQSGAEVKKPGASVKVSCKASGYTFSDYEMHWVRQAPGQGLEWLGA IDPETGGTAYNQKFKGRVTMTGDTSISTAYMELSRLTSDDTAVYYCTGYY DYDSFTYWGQGTLVSVSS 4(GS)x3 linker (SEQ ID No: 8) GGGGSGGGGSGGGGS h2A2 light chain variable domain (VL) (SEQ ID No: 2) DIQMTQSPSSLSTSVGDRVTITCKASQNVDAAVAWYQQKPGKAPKLLIYS ASNRYTGVASRFSGSGSGTDFTFTISSLQSEDLADYFCQQYDIYPYTFGQ GTKLEIK IgG4 hinge domain (SEQ ID No: 9) ESKYGPPCPPCP CD28 transmembrane domain (SEQ ID No: 11) MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB costimulatory domain (SEQ ID No: 12) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3z signaling domain (SEQ ID No: 13) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR T2A ribosomal skipping sequence (SEQ ID No: 14) LEGGGEGRGSLLTCGDVEENPGPR GMCSF signal peptide (SEQ ID No: 7) MLLLVTSLLLCELPHPAFLLIP EGFRt (SEQ ID No: 15) RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTH TPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQH GQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGR ECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCA HYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGL EGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM GMCSF signal peptide (SEQ ID No: 7) MLLLVTSLLLCELPHPAFLLIP hR11 heavy chain variable domain (VH) (SEQ ID No: 3) EVQLVQSGGGLVQPGGSLRLSCAASGSDINDYPISWVRQAPGKGLEWVSF INSGGSTWYASWVKGRFTISRDNAKNSLYLQMNSLRDDDTATYFCARGYS TYYGDFNIWGQGTLVTVSS 4(GS)x3 linker (SEQ ID No: 8) GGGGSGGGGSGGGGS hR11 light chain variable domain (VL) (SEQ ID No: 4) DIVMTQSPSSLSASVGDRVTITCQASQSIDSNLAWFQQKPGKAPKSLIYR ASNLASGVPSKFSGSGSGTDFTLTISSLQREDAATYYCLGGVGNVSYRTS FGGGTKVEIK IgG4 CH2CH3 4/2NQ (SEQ ID No: 10) ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK CD28 transmembrane domain (SEQ ID No: 11) MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB costimulatory domain (SEQ ID No: 12) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3z signaling domain (SEQ ID No: 13) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR T2A ribosomal skipping sequence (SEQ ID No: 14) LEGGGEGRGSLLTCGDVEENPGPR GMCSF signal peptide (SEQ ID No: 7) MLLLVTSLLLCELPHPAFLLIP EGRFt (SEQ ID No: 15) RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTH TPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQH GQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGR ECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCA HYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGL EGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM GMCSF signal peptide (SEQ ID No: 7) MLLLVTSLLLCELPHPAFLLIP hR12 heavy chain variable domain (VH) (SEQ ID No: 5) QVQLVESGGALVQPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIAT IYPSSGKTYYAASVQGRFTISADNAKNTVYLQMNSLTAADTATYFCARDS YADDGALFNIWGQGTLVTVSS 4(GS)x3 linker (SEQ ID No: 8) GGGGSGGGGSGGGGS hR12 light chain variable domain (VL) (SEQ ID No: 6) QLVLTQSPSVSAALGSSAKITCTLSSAHKTDTIDWYQQLAGQAPRYLMYV QSDGSYEKRSGVPDRFSGSSSGADRYLIISSVQADDEADYYCGADYIGGY VFGGGTQLTVG IgG4 hinge domain (SEQ ID No: 9) ESKYGPPCPPCP CD28 transmembrane domain (SEQ ID No: 11) MFWVLVVVGGVLACYSLLVTVAFIIFWV 4-1BB costimulatory domain (SEQ ID No: 12) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3z signaling domain (SEQ ID No: 13) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR T2A ribosomal skipping sequence (SEQ ID No: 14) LEGGGEGRGSLLTCGDVEENPGPR GMCSF signal peptide (SEQ ID No: 7) MLLLVTSLLLCELPHPAFLLIP EGFRt (SEQ ID No: 15) RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTH TPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQH GQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGR ECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCA HYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGL EGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

Additionally, preferred amino acid sequences of ROR1-binding fragments of the non-humanized monoclonal antibodies R11, R12 and 2A2 that can be used as starting sequences for humanization in accordance with the invention are as indicated below:

scFV of the non-humanized 2A2 antibody (SEQ ID No: 16): QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEWIGA IDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTGYY DYDSFTYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSQKIMSTTVGD RVSITCKASQNVDAAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGS GTDFTLTISNMQSEDLADYFCQQYDIYPYTFGGGTKLEIK scFV of the non-humanized R11 antibody (SEQ ID No: 17): QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFI NSGGSTWYASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYY GDFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVMTQTPSSTSGAVGGTV TINCQASQSIDSNLAWFQQKPGQPPTLLIYRASNLASGVPSRFSGSRSGT EYTLTISGVQREDAATYYCLGGVGNVSYRTSFGGGTEVVVK scFV of the non-humanized R12 antibody (SEQ ID No: 18): QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIAT IYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDS YADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAAL GSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPD RFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG VH of the non-humanized 2A2 antibody (SEQ ID No: 19): QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEWIGA IDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTGYY DYDSFTYWGQGTLVTVSA VL of the non-humanized 2A2 antibody (SEQ ID No: 20): DIVMTQSQKIMSTTVGDRVSITCKASQNVDAAVAWYQQKPGQSPKLLIYS ASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYDIYPYTFGG GTKLEIK VH of the non-humanized R11 antibody (SEQ ID No: 21): QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFI NSGGSTWYASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYY GDFNIWGPGTLVTISS VL of the non-humanized R11 antibody (SEQ ID No: 22): ELVMTQTPSSTSGAVGGTVTINCQASQSIDSNLAWFQQKPGQPPTLLIYR ASNLASGVPSRFSGSRSGTEYTLTISGVQREDAATYYCLGGVGNVSYRTS FGGGTEVVVK VH of the non-humanized R12 antibody (SEQ ID No: 23): QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIAT IYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDS YADDGALFNIWGPGTLVTISS VL of the non-humanized R12 antibody (SEQ ID No: 24): ELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQV QSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGY VFGGGTQLTVTG

FURTHER PREFERRED EMBODIMENTS

A preferred embodiment of the humanized CARs of the invention is their application in cellular immunotherapy against malignancies that are associated with the aberrant occurrence of ROR1-expressing cells. Preferably, the CAR modified cell is a CD8+ killer T cell, a CD4+ helper T cell, a naïve T cell, a memory T cell, a central memory T cells, an effector memory T cell, a memory stem T cell, an invariant T cell, an NKT cell, a cytokine induced killer T cell, a gamma/delta T cell, a B lymphocyte, a natural killer cell, a monocyte, a macrophage, a dendritic cell, a granulocyte, or any other mammalian cell type desirable to be used for genetic modification.

A particularly preferred embodiment is the usage use of CARs of the invention with humanized targeting domains originating from the 2A2, R11 or R12 monoclonal antibody as immunotherapeutic agents against ROR1-positive leukemia, mantle cell lymphoma, breast-cancer, lung cancer or any other cancer that expresses ROR1.

Another preferred embodiment is the usage use of CARs of the invention with humanized targeting domains originating from the 2A2, R11 or R12 monoclonal antibody as immunotherapeutic agents for the treatment of obesity.

Another preferred embodiment is the usage use of CARs of the invention with humanized targeting domains originating from the 2A2, R11 or R12 monoclonal antibody as immunotherapeutic agents against ROR1-positive autoimmune or infectious diseases.

Another preferred embodiment is the use of the humanized targeting domains of the invention as component of CARs containing a single costimulatory domain, including but not limited to 4-1BB, CD28, Ox40, ICOS, DAP10 or any other domain that provides costimulation to immune cells.

In another embodiment the humanized targeting domains of the invention may be used as components of a CAR that mediates an inhibitory signal due to the usage of a co-inhibitory signaling domain. Such signaling domains can originate from the co-inhibitory receptors CTLA-4, PD-1, BTLA, LAG3, TIM3 or any other receptor that inhibits immune cell functions.

Another preferred embodiment is the usage of the humanized targeting domains of the invention in a CAR that encompasses a combination of two or more costimulatory or co-inhibitory domains.

In another embodiment, the humanized targeting domains of the invention may be used in a format that is different from the presented scFv format to be included into a CAR construct. As a non-limiting example such CARs may be composed of two different polypeptide chains from which one chain encompasses the variable heavy chain (VH) and one chain encompasses the variable light (VL) chain of the disclosed humanized anti-ROR1 antibodies.

In another preferred embodiment the CAR gene, containing a humanized targeting domain of the invention, is transferred into the desired cells by non-viral transfection methods like electroporation, nucleofection or together with a transposase like Sleeping Beauty, PiggyBac, Frog Prince, Himarl , Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmar1, or any of their respective derivatives with equal, lower and/or higher transposition activity.

In another preferred embodiment the CAR gene encompassing a humanized targeting domain originating of the invention is delivered as a part of a RNA or DNA polynucleotide molecule.

EXAMPLES

The present invention is exemplified by the following non-limiting examples:

Example 1 Preparation and Functional Testing of ROR1-Specific CAR-Modified Human CD8+ and CD4+ T Cells with Humanized Targeting Domains Materials and Methods:

Human Subjects

Blood samples were obtained from healthy donors who provided written informed consent to participate in research protocols approved by the Institutional Review Board of the University of Würzburg (Universitätsklinikum Würzburg, UKW). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque (Sigma, St. Louis, Mo.).

Cell Lines

The 293T, K562, MDA-MB-231 and A549 cell lines were obtained from the American Type Culture Collection. K562-ROR1 were generated by lentiviral transduction with the full-length ROR1-gene. Luciferase expressing lines were derived by lentiviral transduction of the above mentioned cell lines with the firefly (P. pyralis) luciferase (ffluc)-gene. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 U/ml penicillin/streptomycin.

Immunophenotyping

PBMC and T cell lines were stained with one or more of the following conjugated mAb: CD3, CD4, CD8 and matched isotype controls (BD Biosciences, San Jose, Calif.). Transduced T cell lines were stained with biotin-conjugated anti-EGFR antibody (ImClone Systems Incorporated, Branchburg, N.J.) and streptavidin-PE (BD Biosciences, San Jose, Calif.). Staining with 7-AAD (BD Biosciences) was performed for live/dead cell discrimination as directed by the manufacturer. Flow analyses were done on a FACSCanto and data analyzed using FlowJo software (Treestar, Ashland, Oreg.).

Lentiviral Vector Construction, Preparation of Lentivirus, and Generation of CAR-T Cells

The construction of epHIV7 lentiviral vectors containing ROR1-specific CARs with a short or long spacer and a 4-1BB costimulatory domain has been described, see reference [35], which is hereby incorporated by reference in its entirety for all purposes. All CAR constructs encoded a truncated epidermal growth factor receptor (EGFRt; also known as tEGFR), see reference [36], which is hereby incorporated by reference in its entirety for all purposes, downstream of the CAR. The genes were linked by a T2A ribosomal skip element.

CAR/EGFRt and ffluc/eGFP-encoding lentivirus supernatants were produced in 293T cells co-transfected with each of the lentiviral vector plasmids and the packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G using Calphos transfection reagent (Clontech, Mountain View, Calif.). Medium was changed 16 h after transfection, and lentivirus collected after 72 h. CAR-T cells were generated as described [35]. In brief, CD8⁺ or CD4⁺ bulk T cells were sorted from PBMC of healthy donors, activated with anti-CD3/CD28 beads (Life Technologies), and transduced with lentiviral supernatant. Lentiviral transduction was performed on day 2 by spinoculation, and T cells propagated in RPMI-1640 with 10% human serum, GlutaminMAX (Life technologies), 100 U/mL penicillin-streptomycin and 50 U/mL IL-2. Trypan blue staining was performed to quantify viable T cells. After expansion, EGFRt⁺ T cells were enriched and expanded by polyclonal stimulation with the CD3-specific Okt3 antibody and irradiated allogeneic PBMC and EBV-LCL feeder cells.

Cytotoxicity, Cytokine Secretion, and CFSE Proliferation Assays

Target cells stably expressing firefly luciferase were incubated in triplicate at 5×10³ cells/well with effector T cells at various effector to target (E:T) ratios. After a four-hour incubation luciferin substrate was added to the co-culture and the decrease in luminescence signal in wells that contained target cells and T cells, compared to target cells alone, measured using a luminometer (Tecan). Specific lysis was calculated using the standard formula. For analysis of cytokine secretion, 5×10⁴ T cells were plated in triplicate wells with target cells at a ratio of 4:1 and IFN-γ, TNF-α, and IL-2 measured by ELISA (Biolegend) in supernatant removed after a 24-hour incubation. For analysis of proliferation, 5×10⁴ T cells were labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and plated in triplicate wells with target cells at a ratio of 4:1 in CTL medium without exogenous cytokines. After 72 h of incubation, cells were labeled with anti-CD3 or anti-CD4 or anti-CD8 mAb and 7-AAD to exclude dead cells from analysis. Samples were analyzed by flow cytometry and cell division of live T cells assessed by CFSE dilution. The division index was calculated using FlowJo software.

Results Generation, Detection and Enrichment of Humanized ROR1 CAR T Cells

PBMCs from healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation and bulk CD4⁺ or CD8⁺ human T cells were extracted from this cell population using MACS. Directly after isolation the T cells were activated with CD3/28 Dynabeads for two days and then transduced by spinoculation with lentiviral vectors encoding non-humanized or humanized versions of the ROR1-specific CARs at a multiplicity of infection (MOI) of 5. The Dynabeads were removed 4 days after transduction and at day 10 the T cells were enriched for EGFRt-positive cells by labeling with a biotinylated monoclonal αEGFR antibody and MACS with anti-biotin microbeads. After the enrichment, the EGFRt-positive fraction reproducibly accounted for over 90% of total cells except for the hR11 CAR that usually showed a slightly lower percentage of EGFRt-positive cells (FIGS. 4A and 4B).

Cytolytic Activity of Humanized ROR1 CAR T Cells

CAR T cells were generated as described above and their cytolytic activity was assessed in 6 h cytotoxicity assays against the ROR1-positive and ffluc-expressing target cell lines K562-ROR1, MDA-MB-231 and A549 (FIG. 5A). No specific lysis was detected against ROR1-negative K562 controls. The T cells expressing the h2A2 and hR12 CARs exhibited a very potent anti-tumor effect with a high degree of target cell lysis that was dose dependent, with higher E:T ratios causing higher percentages of target cell lysis. A T cell line transduced with a vector control encoding for the EGFRt transduction marker but not for a CAR caused no lysis of any of the target cells. This demonstrates that the CAR itself induced the target cell lysis and also that CAR-independent target cells lysis, that could in principle occur due to allo-recognition by endogenous TCRs, was not detectable in our experiments. In contrast to the h2A2 and hR12 CARs the hR11 CAR was markedly impaired in its cytolytic activity and showed no detectable lysis in the 6 h assay. Notably it caused detectable and specific target cell lysis if the incubation time was increased to e.g. 24 h.

The cytotoxicity assay was repeated under the same conditions with CAR T cells that were generated from n=3 unrelated healthy donors and the ROR1-positive target cells K562-ROR1 and MDA-MB-231 (FIG. 5B). For all donors the lysis observed for the h2A2 and hR12 CARs was consistently strong while the lysis mediated by the hR11 CAR was barely detectable after 6 h incubation time.

Effector Cytokine Secretion Following ROR1-Specific Activation of hROR1 CART Cells

CD4⁺ or CD8⁺ CAR T cells were generated as described above and co-cultured with lethally irradiated ROR1-expressing target cell lines at an E:T ratio of 4:1 for 24 h. After the incubation the cell culture supernatant was collected and analyzed for the presence of the effector cytokines IL-2 and IFN-γ by ELISA. As controls, the cells were co-cultured with ROR1-negative K562 cells or in absence of any target cell (media control). For controlling the general ability of the CAR T cells to produce the effector cytokines of interest the cells were polyclonally stimulated with a combination of the protein kinase C (PKC)/NF-κB-activator phorbol 12-Myristate 13-Acetate (PMA) and the Ca2⁺ ionopohre ionomycin. The assay procedure was repeated for up to n=3 unrelated healthy T cells donors and the measured cytokine concentrations were used for group analysis.

The humanized 2A2 CAR showed a cytokine profile of that was comparable to the non-humanized 2A2 CAR (FIG. 6A). IFN-γ was detected exclusively in samples that included ROR1-positive targets or PMA/Iono and the average concentrations were in the range of 1000-1500 pg/ml for both CD4⁺ and CD8⁺ CAR T cells. IL-2 was also exclusively detected in samples that included ROR1-positive target cells and the average concentrations were in the range of 500-1000 pg/ml for CD4⁺ and CD8⁺ CAR T cells. Surprisingly, the IL-2 secretion of the h2A2 CAR T cells was elevated in comparison to the non-humanized variant for K562-ROR1 targets.

The humanized R11 CAR showed an impaired cytokine secretion as compared to the non-humanized R11 CAR (FIG. 6B). The concentrations of IFN-γ and IL-2 were at background level for the hR11 CAR even in the presence of ROR1-positive target cells while for the non-humanized R11 CAR average concentrations of IFN-γ in the range of 1000 pg/ml for CD4⁺ T cells and 1800 pg/ml for CD8⁺ T cells as well as average IL-2 concentrations ranging from 500-1000 pg/ml were detected. Both CAR T cells lines, either expressing the non-humanized or the humanized R11 CAR, retained the general ability to produce IFN-γ and IL-2 in response to the antigen-unspecific stimulation with PMA/Iono.

The humanized R12 CAR showed a cytokine profile of that was comparable to the non-humanized R12 CAR (FIG. 6C). IFN-γ was detected exclusively in samples that included ROR1-spositive targets or PMA/Iono and the average concentrations were in the range of 1000-1500 pg/ml for both CD4⁺ and CD8⁺ CAR T cells. IL-2 was also exclusively detected in samples that included ROR1-positive target cells and the average concentrations were in the range of 400 pg/ml for CD4⁺ and 500-800 pg/ml for CD8⁺ CAR T cells.

Taken together these results demonstrate that the humanization of the targeting domains of the h2A2 and the hR12 CAR did not diminish the potential of CD4⁺ and CD8⁺ human T cells expressing these CARs to secrete the effector cytokines IFN-γ and IL-2 after encounter of ROR1-positive target cells. The cytokine levels detected were comparable to and in one instance higher than for the non-humanized CARs. The hR11 CAR, in contrast, mediated no detectable secretion of effector cytokines as response to ROR1-positive target cells even though the T cells retained the general ability for the secretion of both cytokines suggesting that the humanization of the hR11 targeting domain was causative for the observed loss of function. These data are evidence for the fact that the use of humanized binding domains in CARs generated by CDR grafting and only marginally decreasing the affinity of the humanized anti-ROR1 antibodies, see reference [31], cannot predict the functionality of CAR T cells comprising said humanized binding domains in comparison to CAR T cells comprising the non-humanized parental binding domains.

Proliferation of Humanized ROR1 CAR T Cells

CD4⁺ or CD8⁺ CAR T cells were generated as described, labeled with CFSE and co-cultured with lethally irradiated ROR1-expressing target cell lines at an E:T ratio of 4:1 for 72 h in the absence of exogenous cytokines. After the incubation time the T cells were collected and analyzed for CFSE dilution by flow cytometry. As a negative control, the CAR T cells were cocultured with ROR1-negative K562 cells and as a positive control in the presence of 50 Ul/ml IL-2.

ROR1-negative K562 caused no proliferation of T cells expressing any of the CAR constructs. T cells expressing a vector construct encoding the EGFRt transduction marker but lacking a CAR sequence showed no proliferation in response to any of the target cells above background proliferation (FIG. 7A-D). This demonstrates that the detected proliferation of the ROR1 CAR-T cells was mediated specifically by the CAR as a response to stimulation by ROR1-expressing cells.

Surprisingly, despite similar cytokine secretion profiles, as determined above, T cells expressing the humanized 2A2 CAR proliferated significantly stronger than T cells expressing the non-humanized 2A2 CAR in response to ROR1-positive target cells (FIG. 7A). Depending on the target cell line, the division indices of the CD4⁺ h2A2 CAR T cells were consistently 2-3 fold higher as compared to the non-humanized 2A2 ROR1 CAR (FIG. 7B). T cells expressing the humanized 2A2 CAR went through a higher number of cell divisions than T cells expressing the non-humanized 2A2 CAR (FIG. 7C). 60% of the h2A2 CAR T cells with MDA-MB-231 target cells went through three or more cell division cycles, for the non-humanized 2A2 CAR this fraction was 20%. Similarly, 51% of the h2A2 CAR T cells with K562-ROR1 target cells went through three or more cell division cycles, for the non-humanized 2A2 CAR this fraction was 18%. In line with the previous observations, 18% of the h2A2 CAR T cells with A549 target cells went through three or more cell division cycles, for the non-humanized 2A2 CAR this fraction was 5%.

T cells expressing the humanized R11 showed a weaker proliferation than T cells expressing the non-humanized R11 CAR but the proliferation was clearly above background level (FIG. 7A). Depending on the target cells, the division indices were reduced by a factor of 1.5-3.5.

The proliferation of T cells expressing the humanized R12 CAR was specific and overall comparable to T cells expressing the non-humanized variant. Significantly higher proliferation levels were detected for T cells expressing the humanized R12 CAR as compared to the non-humanized variant response to MDA-MB-231. The proliferation index was about 2-fold increased as compared to the non-humanized R12 CAR variant. The percentage of T cells that went through 3 or more cell divisions was 37% for the humanized R12 CAR and 20% for the non-humanized R12 CAR.

Taken together these results demonstrate that the humanized variants of the 2A2 and R12 CARs are capable of activating T cell proliferation in response to antigen encounter at a substantially higher level than the non-humanized variants. The humanized R11 on the other hand had a pronounced decrease in proliferative capacity and the proliferation levels were markedly lowered as compared to the non-humanized R11 CAR.

Example 2 Binding of ROR1-Protein by Humanized ROR1 CARs

Materials and methods:

Human Subjects

Blood samples were obtained from healthy donors who provided written informed consent to participate in research protocols approved by the Institutional Review Board of the University of Würzburg (Universitätsklinikum Würzburg, UKW). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque (Sigma, St. Louis, Mo.).

Immunophenotyping

PBMC and T cell lines were stained with one or more of the following conjugated mAb: CD3, CD4, CD8 and matched isotype controls (BD Biosciences, San Jose, Calif.). Transduced T cell lines were stained with biotin-conjugated anti-EGFR antibody (ImClone Systems Incorporated, Branchburg, N.J.) and streptavidin-PE (BD Biosciences, San Jose, Calif.). Staining with 7-AAD (BD Biosciences) was performed for live/dead cell discrimination as directed by the manufacturer. Flow analyses were done on a FACSCanto and data analyzed using FlowJo software (Treestar, Ashland, Oreg.).

Lentiviral Vector Construction, Preparation of Lentivirus, and Generation of CAR-T Cells

The construction of epHIV7 lentiviral vectors containing ROR1-specific CARs with a short or long spacer and a 4-1BB costimulatory domain has been described, see reference [35], which is hereby incorporated by reference in its entirety for all purposes. All CAR constructs encoded a truncated epidermal growth factor receptor (EGFRt; also known as tEGFR), see reference [36], which is hereby incorporated by reference in its entirety for all purposes, downstream of the CAR. The genes were linked by a T2A ribosomal skip element.

CAR/EGFRt and ffluc/eGFP-encoding lentivirus supernatants were produced in 293T cells co-transfected with each of the lentiviral vector plasmids and the packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G using Calphos transfection reagent (Clontech, Mountain View, Calif.). Medium was changed 16 h after transfection, and lentivirus collected after 72 h. CAR-T cells were generated as described [35]. In brief, CD8⁺ or CD4⁺ bulk T cells were sorted from PBMC of healthy donors, activated with anti-CD3/CD28 beads (Life Technologies), and transduced with lentiviral supernatant. Lentiviral transduction was performed on day 2 by spinoculation, and T cells propagated in RPMI-1640 with 10% human serum, GlutaminMAX (ThermoFisher Scientific, MA), 100 U/mL penicillin-streptomycin and 50 U/mL IL-2. Trypan blue staining was performed to quantify viable T cells. After expansion, EGFRt⁺ T cells were enriched and expanded by polyclonal stimulation with the CD3-specific Okt3 antibody and irradiated allogeneic PBMC and EBV-LCL feeder cells.

Binding of ROR1

Recombinant aggregated ROR1 protein was labeled with the AlexaFluor647 labeling kit (ThermoFisher Scientific, MA) and used to stain T cells expressing ROR1 CARs. The T cells were washed once in PBS, 0.25% FCS and then resuspended in the same buffer containing a final concentration of 5.3 μg/ml labeled ROR1 protein and monoclonal αEGFRt antibody. After an incubation time of 15 min the cells were washed with an excess of PBS, 0.25% FCS and analyzed by flow cytometry.

Results:

Compared to their non-humanized counterparts the humanized 2A2 and R12 ROR1 CARs showed significantly stronger binding to the ROR1 protein (FIG. 8). A higher overall percentage of ROR1 protein binding was detected, suggesting a better surface availability and/or binding capability of the humanized CARs. The percentage of T cells with a distinct AlexaFluor647 signal was 62.7% for the non-humanized and 92.1% for the humanized 2A2 CAR. Similarly, the percentage of T cells with a distinct AlexaFluor647 signal was 47.6% for the non-humanized and 79.0% for the humanized R12 CAR.

Further, the percentage of CAR T cells that showed strong ROR1 binding was increased for the humanized 2A2 and R12 CARs and consequently a lesser frequency of weak ROR1-binding was detected for these samples. The percentage of T cells with high AlexaFluor647 signal was 2.61% for the non-humanized and 20.0% for the humanized 2A2 CAR. Similarly, the percentage of T cells with high AlexaFluor647 signal was 0.7% for the non-humanized and 7.68% for the humanized R12 CAR. This accounts for a roughly 10-fold increase in the number of T cells that strongly bind to the ROR1 protein.

The humanized R11 showed low overall ROR1 protein binding with 4.98% of AlexaFluor647-positive T cells as compared to 97.9% for the non-humanized R11 CAR. Similarly, the frequency of T cells with high AlexaFluor647 signal was 0.019% for the humanized R11 and 66.5% for the non-humanized variant.

In summary these data demonstrate that the humanized versions of the 2A2 and the R12 CAR have a stronger binding to the ROR1 antigen than the non-humanized versions. That was unexpected and may provide an explanation for the elevated activity in parts of the assays that were performed for example 1.

Example 3 Regression of Human Jeko-1 Mantle Cell Lymphoma in NOD/SCID/yc−/− (NSG) mice after adoptive immunotherapy with CAR-T cells expressing humanized ROR1 CARs Materials and Methods:

Human Subjects

Blood samples were obtained from healthy donors who provided written informed consent to participate in research protocols approved by the Institutional Review Board of the University of Würzburg (Universitätsklinikum Würzburg, UKW). Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque (Sigma, St. Louis, Mo.).

Cell Lines

The JeKo-1 (wild type), 293T, K562, MDA-MB-231 and A549 cell lines were obtained from the American Type Culture Collection. K562-ROR1 were generated by lentiviral transduction with the full-length ROR1-gene. Luciferase expressing lines were derived by lentiviral transduction of the above mentioned cell lines with the firefly (P. pyralis) luciferase (ffluc)-gene. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 U/ml penicillin/streptomycin.

Immunophenotyping

PBMC and T cell lines were stained with one or more of the following conjugated mAb: CD3, CD4, CD8 and matched isotype controls (BD Biosciences, San Jose, Calif.). Transduced T cell lines were stained with biotin-conjugated anti-EGFR antibody (ImClone Systems Incorporated, Branchburg, N.J.) and streptavidin-PE (BD Biosciences, San Jose, Calif.). Staining with 7-AAD (BD Biosciences) was performed for live/dead cell discrimination as directed by the manufacturer. Flow analyses were done on a FACSCanto and data analyzed using FlowJo software (Treestar, Ashland, Oreg.).

Lentiviral Vector Construction, Preparation of Lentivirus, and Generation of CAR-T Cells and JeKo-1/ffluc Cells

The construction of epHIV7 lentiviral vectors containing ROR1-specific CARs with a short or long spacer and a 4-1BB costimulatory domain has been described, see reference [35], which is hereby incorporated by reference in its entirety for all purposes. All CAR constructs encoded a truncated epidermal growth factor receptor (EGFRt; also known as tEGFR), see reference [36], which is hereby incorporated by reference in its entirety for all purposes, downstream of the CAR. The genes were linked by a T2A ribosomal skip element.

CAR/EGFRt and ffluc/eGFP-encoding lentivirus supernatants were produced in 293T cells co-transfected with each of the lentiviral vector plasmids and the packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G using Calphos transfection reagent (Clontech, Mountain View, Calif.). Medium was changed 16 h after transfection, and lentivirus collected after 72 h. CAR-T cells were generated as described [35]. In brief, CD8⁺ or CD4⁺ bulk T cells were sorted from PBMC of healthy donors, activated with anti-CD3/CD28 beads (Life Technologies), and transduced with lentiviral supernatant. Lentiviral transduction was performed on day 2 by spinoculation, and T cells propagated in RPMI-1640 with 10% human serum, GlutaminMAX (Life technologies), 100 U/mL penicillin-streptomycin and 50 U/mL IL-2. Trypan blue staining was performed to quantify viable T cells. After expansion, EGFRt⁺ T cells were enriched and expanded by polyclonal stimulation with the CD3-specific Okt3 antibody and irradiated allogeneic PBMC and EBV-LCL feeder cells. JeKo-1/ffluc cells were generated by lentiviral transduction with the ffluc/eGFP-encoding lentivirus.

Cytotoxicity, Cytokine Secretion, and CFSE Proliferation Assays

Target cells stably expressing firefly luciferase were incubated in triplicate at 5×10³ cells/well with effector T cells at various effector to target (E:T) ratios. After a four-hour incubation luciferin substrate was added to the co-culture and the decrease in luminescence signal in wells that contained target cells and T cells, compared to target cells alone, measured using a luminometer (Tecan). Specific lysis was calculated using the standard formula. For analysis of cytokine secretion, 5×10⁴ T cells were plated in triplicate wells with target cells at a ratio of 4:1 and IFN-γ, TNF-α, and IL-2 measured by ELISA (Biolegend) in supernatant removed after a 24-hour incubation. For analysis of proliferation, 5×10⁴ T cells were labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and plated in triplicate wells with target cells at a ratio of 4:1 in CTL medium without exogenous cytokines. After 72 h of incubation, cells were labeled with anti-CD3 or anti-CD4 or anti-CD8 mAb and 7-AAD to exclude dead cells from analysis. Samples were analyzed by flow cytometry and cell division of live T cells assessed by CFSE dilution. The division index was calculated using FlowJo software.

Experiments in NOD/SCID/γc−/− (NSG) Mice

The Institutional Animal Care and Use Committee approved all mouse experiments. Six-to 8-week old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained from the Jackson Laboratory or bred in-house. Mice were injected with 0.5×10⁶ JeKo-1/ffluc tumor cells via tail vein and received a subsequent tail vein injection of h2A2 or hR12 ROR1 CAR cells. Both ROR1 CAR T cell lines (h2A2 and hR12) expressed the transduction marker EGFRt. Control T cells expressed only the EGFRt transduction marker.

For bioluminescence imaging of tumor growth, mice received intraperitoneal injections of luciferin substrate (Caliper Life Sciences) resuspended in PBS (15 μg/g body weight). Mice were anesthetized with isoflurane and imaged using an Xenogen IVIS Imaging System (Caliper) 10 minutes after luciferin injection in small binning mode at an acquisition time of 1 s to 1 min to obtain unsaturated images. Luciferase activity was analyzed using Living Image Software (Caliper). The photon flux (radiance) was measured within regions of interest that encompassed the entire body of each individual mouse.

Results:

To assess the in vivo activity of humanized ROR1 CARs, we inoculated cohorts (n=5) of immunodeficient NSG mice with the human, ROR1-expressing mantle cell lymphoma line JeKo-1/ffluc by tail vein injection. 21 days later, when the tumor was disseminated, the mice were treated with a single intravenous dose of hR12 or h2A2 ROR1 CAR T cells. Control mice were treated with T cells only expressing an EGFRt control vector or were left untreated.

We observed rapid tumor regression and improved survival in all of the mice treated with hR12 and h2A2 ROR1 CAR-T cells (response rate 100%). FIG. 9A displays the tumor regression 7 days after T cell transfer as percentage change from baseline radiance. The baseline was measured for each mouse before the treatment on day 21. In contrast to humanized ROR1 CAR-treated mice, the control groups, which were either left untreated or were treated with T cells expressing the EGFRt control vector, showed continued tumor growth in all of the mice (response rate 0%). The average radiance 7 days after CAR T cell treatment was 2*10⁴ p/sec/cm²/sr for hR12 and 1*10⁵ for h2A2. The average radiance of the control groups was 6*10⁶ for untreated mice and 1*10⁶ for mice treated with the EGFRt-only vector control.

Humanized ROR1 CAR T cells engrafted and persisted in the mice and were detectable in spleen and bone marrow of the mice until the end of the experiment. FIG. 9B shows representative flow cytometry data from bone marrow samples of one mouse from the hR12 cohort and one mouse of the h2A2 cohort at day 56. CD4 and CD8 T cells were detectable, and expressed the humanized ROR1 CAR. We confirmed the presence of humanized ROR1 CAR T cells in organ samples; the proportion of humanized ROR1 CAR T cells in the total cells varied between individual mice but was generally in the range of 1-10%.

In summary, these data demonstrate that the h2A2 and the hR12 CARs conferred a strong and specific anti-tumor activity in vivo.

INDUSTRIAL APPLICABILITY

The CARs, combinations of CARs, the recombinant mammalian cells and the methods and medical uses according to the invention are industrially applicable. For example, they can be used as, or for the production of, pharmaceutical products.

REFERENCES

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1-57. (canceled)
 58. A ROR1-specific CAR comprising a humanized targeting domain capable of binding to ROR1, wherein the humanized targeting domain comprises, preferably in an N- to C-terminal order: a) an antibody heavy chain variable domain amino acid sequence selected from the group consisting of: a1) the amino acid sequence of SEQ ID No: 1 or an amino acid sequence at least 90% identical thereto; a2) the amino acid sequence of SEQ ID No: 3 or an amino acid sequence at least 90% identical thereto; or a3) the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 90% identical thereto; and b) an antibody light chain variable domain amino acid sequence selected from the group consisting of: b1) the amino acid sequence of SEQ ID No: 2 or an amino acid sequence at least 90% identical thereto; b2) the amino acid sequence of SEQ ID No: 4 or an amino acid sequence at least 90% identical thereto; or b3) the amino acid sequence of SEQ ID No: 6 or an amino acid sequence at least 90% identical thereto.
 59. The ROR1-specific CAR according to claim 58, wherein the humanized targeting domain consists of the following sequences in an N- to C-terminal order: I) the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 1, an amino acid linker sequence which is preferably the amino acid sequence of SEQ ID No: 8, and the antibody light chain variable domain amino acid sequence of SEQ ID No: 2; II) the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 3, an amino acid linker sequence which is preferably the amino acid sequence of SEQ ID No: 8, and the antibody light chain variable domain amino acid sequence of SEQ ID No: 4; or III) the antibody heavy chain variable domain amino acid sequence of SEQ ID No: 5, an amino acid linker sequence which is preferably the amino acid sequence of SEQ ID No: 8, and the antibody light chain variable domain amino acid sequence of SEQ ID No:
 6. 60. A combination of CARs comprising at least a first and a second CAR, the combination being ROR1-specific, wherein said first and said second CAR are present on different polypeptide chains, and wherein: c) said first CAR comprises a first humanized targeting domain comprising an antibody heavy chain variable domain amino acid sequence selected from the group consisting of: c1) the amino acid sequence of SEQ ID No: 1 or an amino acid sequence at least 90% identical thereto; c2) the amino acid sequence of SEQ ID No: 3 or an amino acid sequence at least 90% identical thereto; or c3) the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 90% identical thereto; and d) said second CAR comprises a second humanized targeting domain comprising an antibody light chain variable domain amino acid sequence selected from the group consisting of: d1) the amino acid sequence of SEQ ID No: 2 or an amino acid sequence at least 90% identical thereto; d2) the amino acid sequence of SEQ ID No: 4 or an amino acid sequence at least 90% identical thereto; or d3) the amino acid sequence of SEQ ID No: 6 or an amino acid sequence at least 90% identical thereto.
 61. The ROR1-specific CAR according to claim 58, wherein the CAR further comprises a costimulatory domain capable of mediating costimulation to immune cells, wherein at least the first or the second CAR of the combination, preferably at least the first and the second CAR of the combination, most preferably all of the CARs of the combination, further comprise a costimulatory domain capable of mediating costimulation to immune cells, optionally wherein said costimulatory domain is from 4-1BB, CD28, Ox40, ICOS or DAP10, further optionally wherein said costimulatory domain has the amino acid sequence of SEQ ID No:
 12. 62. The ROR1-specific CAR according to claim 58, further comprising a transmembrane polypeptide, wherein at least the first or the second CAR of the combination, preferably at least the first and the second CAR of the combination, most preferably all of the CARs of the combination, further comprise a transmembrane polypeptide, optionally wherein said transmembrane polypeptide is a transmembrane domain from CD4, CD8 or CD28, and optionally wherein said transmembrane domain has the amino acid sequence of SEQ ID No:
 11. 63. The ROR1-specific CAR according to claim 58, further comprising a CAR spacer domain, wherein at least the first or the second CAR of the combination, preferably at least the first and the second CAR of the combination, most preferably all of the CARs of the combination, further comprise a CAR spacer domain, optionally wherein said CAR spacer domain is from CD4, CD8, an FC-receptor, an immunoglobulin, or an antibody, and optionally wherein said CAR spacer domain has the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 10, preferably the amino acid sequence of SEQ ID No:
 9. 64. The ROR1-specific CAR according to claim 58, further comprising a suicide gene product that allows the selective killing of CAR T cells, wherein at least the first or the second CAR of the combination further comprises a suicide gene product that allows the selective killing of CAR T cells, optionally wherein said suicide gene product is iCasp9 or HSV-TK.
 65. The ROR1-specific CAR according to claim 58, comprising, in an N- to C-terminal order: i) a signal peptide for direction into the endoplasmic reticulum, the signal peptide preferably having the amino acid sequence of SEQ ID No: 7; ii) a humanized targeting domain; iii) the CAR spacer domain; iv) a transmembrane polypeptide; v) a costimulatory domain; vi) a CD3z signaling domain preferably having the amino acid sequence of SEQ ID No: 13; and optionally further comprising: vii) a T2A ribosomal skipping sequence preferably having the amino acid sequence of SEQ ID No: 14; viii) a signal peptide for direction into the endoplasmic reticulum, the signal peptide preferably having the amino acid sequence of SEQ ID No: 7; and ix) a detectable marker protein sequence preferably having the EGFRt amino acid sequence of SEQ ID No: 15, optionally wherein the ROR1-specific CAR consists of said components i) to ix).
 66. A polynucleotide encoding the ROR1-specific CAR according to claim
 58. 67. A recombinant mammalian cell expressing at least one ROR1-specific CAR according to claim 58, optionally wherein said cell is an immune cell, further optionally wherein said cell is a lymphocyte, optionally wherein said lymphocyte is a B lymphocyte or T lymphocyte, or optionally wherein said cell is a CD8+ killer T cell, a CD4+ helper T cell, a naive T cell, a memory T cell, a central memory T cell, an effector memory T cell, a memory stem T cell, an invariant T cell, an NKT cell, a cytokine induced killer T cell, a g/d T cell, a natural killer cell, a monocyte, a macrophage, a dendritic cell, or a granulocyte, optionally wherein the cell is a human cell.
 68. The recombinant mammalian cell according to claim 67, wherein the recombinant mammalian cell is capable of at least one cell division when cocultured with lethally irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for 72 h in the absence of exogenous cytokines, optionally wherein the recombinant mammalian cell is capable of at least two cell divisions when cocultured with lethally irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for 72 h in the absence of exogenous cytokines, optionally wherein the recombinant mammalian cell is capable of at least three cell divisions when cocultured with lethally irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for 72 h in the absence of exogenous cytokines, optionally wherein the recombinant mammalian cell is capable of at least four cell divisions when cocultured with lethally irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for 72 h in the absence of exogenous cytokines, optionally wherein the recombinant mammalian cell is capable of at least five cell divisions when cocultured with lethally irradiated ROR1-expressing target cells at an E:T ratio of 4:1 for 72 h in the absence of exogenous cytokines.
 69. The recombinant mammalian cell according to claim 67, wherein the ROR1-specific CAR or the ROR1-specific combination of CARs is capable of binding to ROR1 with higher binding affinity than a respective recombinant mammalian control cell expressing a respective ROR1-specific CAR or a respective ROR1-specific combination of CARs where none of the targeting domains is humanized, and wherein said binding affinity is binding affinity to fluorescently labelled recombinant aggregated ROR1 as assessed by flow cytometry analysis.
 70. A method for producing a recombinant mammalian cell according to claim 67, the method comprising the steps of: (a) providing a mammalian cell; (b) introducing into said mammalian cell of step (a) at least one polynucleotide encoding said at least one ROR1-specific CAR or said combination of CARs; and (c) expressing said at least one ROR1-specific CAR or said combination of CARs from said at least one polynucleotide in said cell; thereby obtaining said recombinant mammalian cell, optionally: I) wherein the method further comprises coculturing the recombinant mammalian cell with lethally irradiated ROR1-expressing target cells at a ratio of 4:1 for 72 h in the absence of exogenous cytokines, optionally wherein at least 1%, preferably at least 2%, more preferably at least 3%, and still more preferably at least 4% of the recombinant mammalian cells undergo at least 5 cell divisions during the coculturing; II) wherein said method is an in vitro method; III) wherein said mammalian cells of step (a) are cells obtained from donors; and/or IV) wherein said mammalian cells of step (a) are cells obtained from patients.
 71. A recombinant mammalian cell according to claim 67, for use in medicine.
 72. A recombinant mammalian cell according to claim 67, for use in a method for treating a cancer that expresses ROR1 in a patient, optionally wherein the cancer is ROR1-positive leukemia, mantle cell lymphoma, breast-cancer or lung cancer.
 73. A method for treating a patient in need thereof, the method comprising administering a recombinant mammalian cell according to claim 67 to said patient, optionally wherein the method is for treating a cancer that expresses ROR1, optionally wherein the cancer is ROR1-positive leukemia, mantle cell lymphoma, breast-cancer or lung cancer. 